The bacteria of the family Anaplasmataceae are obligate intracellular parasites. As such, these microorganisms are often difficult to grow and the diseases they cause are difficult to diagnose. Because of the difficulty of growing these microorganisms, large-scale preparation of vaccine antigens is costly and sometimes impossible. The bacteria of the Anaplasmataceae family are pathogenic agents of vector-transmitted diseases of human and animals. They are mostly transmitted by invertebrate vectors such as ticks. Within the family Anaplasmataceae, the microorganisms of the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachieae are causative agents of vector-transmitted diseases and are difficult to grow, especially on a large-scale. Bacterial species belonging to the genus Rickettsieae are not included within the family Anaplasmataceae.
Anaplasmosis is a major tick-borne disease of cattle endemic in the United States. The causative agent, Anaplasma marginals, invades and multiplies in erythrocytes of cattle, causing mild to severe anemia. Annual mortality and morbidity due to anaplasmosis impacting beef cattle herds has caused millions of dollars worth of damage due to, for example, weight loss during acute infections and increased veterinary costs. See e.g., Palmer in Veterinary Protozoan and Hemoparasite Vaccines, J. G. Wright (Ed.) CRC Press Inc., Boca Raton, Fla. 1989.
Anaplasma phagocytophilum causes tick borne fever in sheep, cattle, and bison, and is vectored by I. ricinis. See U.S. Pat. No. 6,284,238, which is fully incorporated by reference herein. In contrast to A. marginals which infects red blood cells, A. phagocytophilum infects granulocytes.
Ehrlichia bacteria are closely related to Anaplasma species. Those species for which a biological vector is known are transmitted by ticks, such as for E. canis. While Anaplasma phagocytophilum prefers to infect granulocytes in its mammalian host, Ehrlichia canis prefers to infect mononuclear white blood cells. Typically, Anaplasma and Ehrlichia are contained within membrane-bound vacuoles of their respective host cells.
The first Ehrlichia species recognized was E. canis. It occurs in all areas of the world where the vector tick, Rhipicephalus sanguineus (the brown dog tick), lives. The disease it causes is sometimes called canine tropical pancytopenia. It is a problem especially in all warm areas of the world, e.g., the southern U.S., Central and South America, the Mediterranean, and South Asia. Ehrlichia canis may be cultivated in the dog cell line DH82 as well as human-dog hybrid cell lines (See Rikihisa, Y. 1991. Clinical Microbiology Reviews, 4:286). Various strains of E. canis are listed in U.S. Patent Application No. 2006/0188524, which is herein incorporated by reference in its entirety. Ehrlichia chaffeensis is associated with human ehrlichiosis. See Maeda, K. et al., 1987, N. Eng. J. Med. 316:853; Dawson, J. E. et al., 1991, J. Clin. Microbiol. 29:2741. E. chaffeensis can also be cultured in DH82 cells.
Neorickettsia bacteria are closely related to Ehrlichia and Anaplasma bacteria. Neorickettsia species include N. risticii and N. sennetsu (both of which were previously classified in the genus Ehrlichia. N. risticii is the causative agent of Potomac horse fever. This disease is known to occur in North America, France and India. N. risticii may be grown in macrophage-monocyte cell lines such as P388D1, T-84 and U937. N. sennetsu is the causative agent of Sennetsu erlichiosis of humans. N. sennetsu grows in murine and human cell lines such P388D, L929, and HeLa.
Infections with bacterial organisms of the Anaplasmataceae family may be diagnosed by direct microscopic examination and/or serodiagnosis. Treatment with antibiotics may be effective. Equine vaccines to protect against N. risticii are commercially available. See Compendium of Veterinary Products, 6th ed., Aurora Arrioja, ed., North American Compendiums, Ltd., Port Huron, Mich. (2001). At least one cattle vaccine to protect against anaplasmosis is also commercially available. See Id. However, vaccines have not been manufactured and sold for large scale prevention of other diseases caused by bacterial organisms such as, for example, E. canis. Presently, it is believed that there are no commercial vaccines for Ehrlichia canis. 
Efforts have been made to grow certain Rickettsiale organisms in a variety of host cells. U.S. Pat. No. 5,192,679, which is herein incorporated by reference in its entirety, relates to continuous propagation of E. canis in a canine monocyte macrophage cell line DH82 in an in vitro medium that supports the growth of DH82 cells. U.S. Published Application No. 2005/0202046, which is herein incorporated by reference in its entirety, relates to E. canis vaccines where the E. canis was cultured in DH82 cells. U.S. Pat. No. 5,401,656, which is herein incorporated by reference in its entirety, relates to propagation of E. chaffeensis and E. canis in an immortalized human endothelial cell line. U.S. Pat. No. 5,869,335, which is herein incorporated by reference in its entirety, relates to culturing certain Rickettsiale bacteria in Ixodes scapularis cell lines. U.S. Pat. No. 5,989,848, which is herein incorporated by reference in its entirety, relates to growth of certain Ehrlichial species on an immortalized human endothelial cell line. U.S. Pat. No. 3,616,202, which is herein incorporated by reference in its entirety, relates to growth of Anaplasma marginale in a rabbit bone marrow tissue culture. U.S. Published Patent Application No. 2006/0057699, which is herein incorporated by reference in its entirety, relates to growth of certain Anaplasma species in mammalian cells. U.S. Published Patent Application No. 2003/0003508, which is herein incorporated by reference in its entirety, relates to culturing Rickettsia pulicis bacterium on a Xenopus laevis cell line. U.S. Pat. Nos. 5,955,359 and 5,976,860, which are herein incorporated by reference in their entirety, relate to culturing certain bacterial species belonging to the Rickettsiales order in certain mammalian cell lines. U.S. Pat. No. 5,877,159, which is herein incorporated by reference in its entirety, relates to methods for introducing and expressing genes in animal cells using certain live invasive bacterial vectors.
A thesis discussing immunization of dogs against canine ehrlichiosis using inactivated Ehrlichia canis organisms has been submitted. Sunita Mahan, Immunisation of German shepherd dogs against canine ehrlichiosis using inactivated Ehrlichia canis organisms, thesis submitted to the Faculty of Veterinary Science at the University of Zimbabwe (May 1997). This thesis discusses use of a β-propiolactone inactivated E. canis organisms in combination with Quill A.
Because growth of bacterial species belonging to the Anaplasmataceae family in host cells has met with only limited success and apparently has not translated into an abundant supply of vaccines, there remains a general need to develop culturing systems for growing such bacterial species to facilitate the study of these pathogenic microorganisms and for the development of vaccines to guard against the diseases they cause. There is also a need to develop a large scale culturing system for preparation of large amounts of antigen from such microorganisms for use in diagnostics and vaccines.